PROJECT SUMMARY/ABSTRACT The upper brainstem is necessary for conscious wakefulness, and lesions dividing this area from the forebrain result in deep coma. However, the neurons originating this ascending arousal system and their forebrain targets necessary for maintaining conscious wakefulness have never been identified. Currently, our ability to diagnose, prognosticate, and ultimately treat patients with disorders of consciousness is constrained by a lack of information regarding the precise location, genetic identity, and connections of these arousal-promoting neurons. We recently analyzed human brainstem lesions and identified an focal area in the upper brainstem that is injured specifically and commonly in patients presenting in coma. This coma-specific area is in the region of the parabrachial nucleus (PB), a site in which experimental lesions in rats produce deep coma. We also found that this coma-specific region exhibits strong and specific ?connectivity? with the frontoinsular cortex (FI) in resting-state functional connectivity MRI (rs-fcMRI) analysis in normal human subjects. In rodents, a subpopulation of PB neurons projects directly to a homologous cortical area (agranular insular cortex), so we hypothesized that PB neurons with direct projections to the frontoinsular cortex (PB-FI) are a core component of the ascending arousal system. To test this hypothesis, we will use connectivity-based mouse genetic tools, accessing PB-FI neurons by injecting AAV6 ?retro-Cre? into the insular cortex and then Cre-dependent vectors in PB. This approach allows us to selectively characterize and manipulate PB-FI neurons without altering the myriad other homeostatic functions mediated by other PB neurons. We will use this approach first to identify the full range of efferent projections of PB-FI neurons, searching for any axon collaterals to subcortical regions that may sustain arousal in parallel with FI. We will then use fiber photometry to measure the activity of PB-FI neurons from calcium transients in awake, behaving mice, and correlate their activity with concurrent behavioral state using continuous video-EEG. Finally, we will activate and ablate PB-FI neurons, testing our hypotheses that they produce wakefulness, and that their absence will result in coma or hypersomnolence. Information about their axon collaterals and activity patterns will enable optogenetic stimulation and inhibition of the fiber terminals of PB-FI neurons in target- and state-specific tests of the role of each projection of PB-FI neurons in maintaining a waking state. Ultimately, these experiments will shed much-needed light on a key, unanswered question in clinical neurology: which brainstem neurons and connections mediate basic, conscious wakefulness?